Abigail Ulloa's DNA Polymerase (A12) Aptamer Project (2016)

Aptamer Selection against A12 DNA Polymerase for use in Hot Start PCR

Introduction/Background

Since its conception in the 1980’s, Polymerase Chain Reaction (PCR) has become an extremely common and valuable lab technique due to its ability to rapidly replicate specific sequences of DNA from a given ‘template’ strand (McPherson and Møller, 2007). PCR works by cycling through several steps performed at different temperatures that denature DNA, allow primers to anneal to the template strands, then permit DNA polymerase to replicate the desired sequence (Figure 2 ) (www.ncbi.nlm.nhi.gov). A thermostable DNA polymerase is necessary to perform PCR; one that is commonly used is Taq, taken from the bacteria Thermus aquaticus (Chien et al., 1978). Although conventional PCR is effective, it still has disadvantages— the greatest of these being the production and amplification of nonspecific PCR products due to nonspecific priming events such as random priming of the template sequence (mispriming) or the priming of one primer onto another: a phenomenon referred to as ‘primer dimers’ (Brownie et al., 1997). These random DNA replications consume the limited amount of primers and nucleosides supplied for the reaction, producing a final product with up to 50x decrease in accuracy- particularly after a large number of rounds of PCR (Wang and Wu, 1994). In order for the synthesis of undesirable DNA sequences to be prevented, the activity of the DNA polymerase must be inhibited below the annealing temperature of the primers. The focus of this study is the A12 DNA polymerase heteroduplex (molecular weight: 91.084 kDa, pI: 8.12) which was purified at the Ellington Lab at The University of Texas at Austin. A12 was created from a recombination of genes taken from the DNA polymerases of the Thermococcus kodakaraensis (KOD) and Pyrococcus furiosus (Pfu) bacteria, which can process replication more quickly and hold larger elongation rates than other DNA polymerases, respectively. A12 DNA Polymerase has the ability to produce a large amount of PCR product in a very short amount of time. One way to inhibit DNA polymerase is through the use of an aptamer: an oligonucleotide sequence that binds tightly and specifically to a target (Figure 3) (Radom et al., 2013). Aptamers are analogous to antibodies, and can be selected to bind very specifically to a target and are thus very versatile. Aptamers are thermally stable, cost-effective, and can be used for a variety of drug delivery, diagnostic and therapeutic purposes. The selected aptamer would optimally inhibit A12 polymerase below the annealing temperature of the primers and denature at a higher temperature to prevent nonspecific priming events from being replicated while allowing for normal PCR function, resulting in a ‘Hot Start’ PCR method (Paul et al., 2010). Previously, an aptamer was found that serves a similar purpose for use with Taq DNA Polymerase. This aptamer inhibits Taq at temperatures below 45°C (Noma et al., 2006). The SELEX method (Figure 4) (Stoltenburg et al., 2007) was employed in finding an aptamer to inhibit A12. Aptamer selection begins with a large and varied pool of RNA sequences containing anywhere from 1012 to 1014 different sequences of RNA. In this case, the N71 pool was used. The sequences in this pool contain a randomized region of nucleotides that is 71 base pairs long and is flanked by known sequences that serve as primer binding sites (Bell et al., 1998). The beads used for this selection were PureProteome Nickel Magnetic Beads. These beads bind targets with a polyhistidine protein tag. Selection was performed in A12 storage buffer- created specifically to be used with A12 DNA polymerase and consists of 5 mM Tris-HCl, 1 mM KCl, 1 mM (NH4)2SO4, 0.2 mM MgSO4, 0.1% Triton-X 100, and 0.001% BSA. A12 is negatively charged within this buffer, which causes it to repel negatively charged DNA. The salts in the buffer serve as a positive bridge between them, allowing RNA sequences to bind to the target. In bead-based selection, the target is attached to the beads, which are then exposed to the pool. Some of the sequences in the pool will bind. In one round of selection, the unbound sequences are removed and the bound sequences are eluted, reverse transcribed, amplified by PCR and transcribed back into RNA. The RNA that is recovered at the end of one round of selection is used as the pool for the subsequent round. Multiple rounds are performed with increasing stringency to narrow down the amount of sequences that bind to the target and find the sequence that best binds. A12’s naturally binds nucleotides, making it an ideal target for aptamer selection. Currently, eight rounds of selection have been performed. A binding assay was conducted to assess the binding affinity of the pool to the target. The binding assay results were inconclusive, and did not indicate that a selection was occurring. Sequencing is being performed to assess the diversity of the selected pool and determine whether more rounds of selection need to be conducted to find the sequences that best bind to A12 DNA Polymerase.